Project Summary/Abstract Patients with indwelling urinary catheters have nearly double the mortality rate compared to non-catheterized patients due to the propensity for adverse outcomes, including functional decline, increased length of stay in hospital settings, catheter-associated urinary tract infections (CAUTI), and bacteremia. Catheterization also increases the likelihood of colonization by multidrug-resistant organisms, and antibiotic resistance is increasing at an alarming rate, making it imperative to identify novel, non-antibiotic treatments for CAUTI and associated complications. The Gram-negative bacterium Proteus mirabilis is a predominant cause of CAUTI, particularly during long-term catheterization. CAUTI is also frequently polymicrobial, which can increase risk of severe disease and bacteremia. There is, however, a fundamental gap in knowledge regarding conserved targets for treating or preventing disease due to P. mirabilis, particularly during polymicrobial infection. Our preliminary data clearly demonstrates that the presence of other bacterial species during infection dramatically impacts the genes required by P. mirabilis for colonization and persistence during CAUTI, especially the metabolic pathways that are favored by the bacterium for growth and the defense mechanisms that are used to evade host antimicrobial responses. However, we have uncovered a set of 217 genes encoded by P. mirabilis that provide a fitness advantage during single-species infection and polymicrobial infection with another Gram-negative bacterium, Providencia stuartii. Fifty-seven percent of these genes (123/217) are highly conserved in all 106 publically- available P. mirabilis genomes. The central hypothesis of this proposal is that P. mirabilis encodes a core set of conserved genes that are critical for establishing CAUTI, regardless of which other bacterial species are present. We further anticipate that a subset of these factors will be ideal targets for treatment or prevention of disease. This hypothesis will be tested through three concurrent specific aims. In Aim 1, we will utilize genome- wide transposon insertion-site sequencing to uncover the impact of co-colonization by two common uropathogens (Escherichia coli and Enterococcus faecalis) on the genes required by P. mirabilis to colonize the catheterized urinary tract, including which of the 123 conserved core fitness factors remain important for establishing infection and whether they are expressed during human CAUTI. In Aims 2 and 3, we will explore the importance of two pathways that are well-represented in the current set of core fitness factors (amino acid metabolism and peroxide detoxification) to P. mirabilis pathogenicity. This includes broad characterization of amino acid availability within the murine urinary tract, with or without an indwelling catheter, and the amino acid preferences of 5 uropathogens, as well as production of reactive oxygen species by these uropathogens and by neutrophils in response to the uropathogens. The knowledge gained herein will uncover unique challenges that the catheterized urinary tract presents to invading uropathogens, as well as novel, conserved, clinically-relevant targets for treating or preventing infections due to these prevalent and increasingly drug-resistant bacteria.